In many fields of endeavor it is important to be able to determine whether or not materials are contaminated with bacteria or the like. Examples of such fields are the medical field, the food processing industry, the pharmaceutical industry, the cosmetics industry, the field of public health, and interplanetary space exploration.
In the past, it has been a standard practice to place a sample of a material to be tested for the presence of bacteria in an appropriate growth medium or a Petri dish and to make visual observations of the resulting microbial growth, if any. Not only are such culturing methods slow and laborious, but because they depend on the subjective judgment of individual human observers, the result obtained is not uniformly reliable.
Techniques have also been developed for detection of bacteria which involve incubating a sample of material to be tested in a closed container with a radioactive isotope labeled culture medium and monitoring the atmosphere in the container above the medium to determine whether or not radioactive gases are produced. A radiometric system of this type is disclosed in U.S. Pat. Nos. 3,676,679 and No. 3,935,073. Radiometric systems are rapid and reliable, but they suffer from a number of disadvantages. In the first place, radioactively labeled materials are not inexpensive and require special handling during storage, use and disposal. Moreover, although the levels of radioactivity encountered in using such systems are very low, prospective users may be deterred by personal fears of radioactivity. The use of radioactive isotopes in instrumental systems has generally been resorted to in order to facilitate detection of minute quantities of metabolic product gases thereby to detect rapidly the presence of bacteria.
Wilkins, U.S. Pat. No. 3,907,646, discloses a method for bacteria detection and quantification based on measurement of an increase in the pressure of the culture gas above a liquid culture medium in a sealed vial. Although this method is simple, it appears to be relatively insensitive and does not give faster detection than is achieved by visual observation of the turbidity of the medium.
It has long been known that many bacteria consume oxygen, but it has been thought that no vacuum would be produced because the oxygen was replaced by evolved carbon dioxide. The comment of Arthur is typical:
"In a closed system, as bacteria utilize oxygen and create carbon dioxide, there is no effective change in pressure." (U.S. Pat. No. 3,740,320, Col. 3, 11. 28-29)
Thus where it has been attempted to measure the consumption of oxygen by known microorganisms, either with a Warburg Apparatus or in some other complex system, a carbon dioxide absorber has invariably been included in the system to capture all evolved carbon dioxide thereby to prevent the evolved carbon dioxide from obliterating the oxygen consumption; see Poepel, U.S. Pat. No. 3,282,803; Arthur, U.S. Pat. No. 3,740,320 and Umbreit, Manometric and Biochemical Techniques, Burgess Publishing Company, Minneapolis, 1972.
In the introduction to his patent on radiometric petroleum prospecting, U.S. Pat. No. 2,777,799, Davis cursorily mentions attempts at petroleum prospecting by incubating soil samples in an atmosphere containing gaseous hydrocarbon and measuring the decrease of the pressure of the atmosphere due to consumption of the hydrocarbon by hydrocarbon consuming bacteria found in earth formations near petroleum deposits. Such attempts are described as unsatisfactory because pressure decreases occur for reasons other than consumption of hydrocarbon by the desired bacteria, and Davis teaches instead a radiometric assay technique.
Attempts to avoid radiometric techniques for bacterial detection have often been complicated, unwieldy and cumbersome like the indirect measurement of oxygen production from H.sub.2 O.sub.2 by the enzyme catalase disclosed in Groves, U.S. Pat. No. 3,838,034.
There is a requirement, especially in medical and industrial applications, for a simple and very rapid non-radioactive method for the detection and quantification of the microorganisms in a test sample. Speed is of the essence since faster detection, at least in the medical field, allows faster application of appropriate treatment. Simplicity, reliability and low cost are also of paramount importance. These advantages may best be obtained in automated instrumental systems. Thus, there exists a need for a rapid, non-radioactive instrumental system for detecting bacteria.
Accordingly, it is an object of the present invention to provide a rapid method for detecting the presence or absence of bacteria.
Another object of the invention is to provide a method for rapidly detecting the presence or absence of bacteria which uses comparatively inexpensive materials.
It is a further object of the present invention to provide an instrumental method for detecting the presence or absence of bacteria which is not subject to the vagaries of subjective human observations.
Another object of the present invention is to provide an instrumental system for detecting the presence or absence of bacteria which avoids the use of radioactive materials.
Another object is a method for rapid detection of microorganisms using a method that is simple, easy to use and can handle a plurality of samples.
A further object of the present invention is to provide an instrumental method for measuring microorganism growth that can be scanned automatically and electronically to detect such growth.
It is likewise an object of this invention to provide a method for detecting bacteria by measuring vacuum production by microorganisms when grown in a culture medium in a sealed vial.
An additional object of the invention is to provide a method of quantifying the number of oxygen consuming microorganisms in a test sample.
Further objects of the invention will be apparent from a consideration of the following description.